The Burkitt's lymphoma receptor 1 (BLR1) gene—also known as Chemokine (C-X-C motif) receptor 5 (CXCR5)—was identified initially in Burkitt lymphoma cells has been the first member of the superfamily of G-protein-coupled receptors with a lymphocyte specific expression pattern. BLR1 shows significant relationship to receptors for chemokines (IL-8, MIP-1 beta) and neuropeptides. SCID mice in which mature B cell development is severely impaired exhibit a strongly reduced level of Blr1-specific RNA in the spleen. The analysis of murine lymphoid tumor cell lines representing distinct stages of the B cell lineage reveals elevated expression of Blr1 in B cell lymphomas but not in pre-B lymphomas or plasmacytomas. Murine BLR1 may represent a cytokine/neuropeptide receptor exerting regulatory functions on recirculating mature B lymphocytes (Kaiser E, Förster R, Wolf I, Ebensperger C, Kuehl W M, Lipp M. The G protein-coupled receptor BLR1 is involved in murine B cell differentiation and is also expressed in neuronal tissues. Eur J. Immunol. 1993 October; 23(10):2532-9; Förster R, Wolf I, Kaiser E, Lipp M. Selective expression of the murine homologue of the G-protein-coupled receptor BLR1 in B cell differentiation, B cell neoplasia and defined areas of the cerebellum. Cell Mol Biol (Noisy-le-grand). 1994 May; 40(3):381-7).
Database entry ABK41953 discloses the amino acid sequence of human Burkitt lymphoma receptor 1, GTP binding protein (chemokine (C-X-C motif) receptor 5), database entry EF064770 discloses the nucleotide sequence of human Burkitt lymphoma receptor 1, GTP binding protein (chemokine (C-X-C motif) receptor 5) (BLR1) gene.
WO 99/28468 and U.S. Pat. No. 6,110,695 describe methods and compositions for identifying agents which modulate the interaction of the chemokine receptor Burkitt's Lymphoma Receptor 1 (BLR1) with its ligand, B Lymphocyte Chemoattractant (BLC), and for modulating the interaction of BLR1 and BLC polypeptides. The methods for identifying BLR1:BLC modulators are described as finding particular application in commercial drug screens.
Specific patterns of chemokine receptor expression ensure an orchestrated migration of leukocytes throughout the body, where immune cells home to their target tissues both under steady-state and inflammatory conditions. The chemokine receptor CCR6 is widely expressed on human blood and tissue leukocytes, among them subsets of dendritic cells, CD45RO+ effector/memory T cells, CD25high regulatory T cells (Treg), naive and memory B cells, NKT cells and NK cells (Berahovich R D, Lai N L, Wei Z, Lanier L L, Schall T J. Evidence for NK cell subsets based on chemokine receptor expression. J Immunol. 2006; 177:7833-7840.). On memory T cells, CCR6 is expressed on a population of auto-reactive IL-10-producing cells (Dieu M C, Vanbervliet B, Vicari A, et al. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med. 1998; 188:373-386.) and on the majority of skin- and mucosa-homing cells. Several studies have further shown that CCR6 is consistently expressed on inflammatory IL-17-producing CD4+ T cells (Hirota K, Yoshitomi H, Hashimoto M, et al. Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model. J Exp Med. 2007; 204:2803-2812.) which are involved in a wide array of adverse inflammatory diseases, such as rheumatoid arthritis, psoriatic disease, inflammatory bowel disease or encephalopathies in mice and men (Hirota K, Yoshitomi H, Hashimoto M, et al. Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model. J Exp Med. 2007; 204:2803-2812. Ruth J H, Shahrara S, Park C C, et al. Role of macrophage inflammatory protein-3alpha and its ligand CCR6 in rheumatoid arthritis. Lab Invest. 2003; 83:579-588. Homey B, Dieu-Nosjean M C, Wiesenborn A, et al. Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis. J Immunol. 2000; 164:6621-6632. Hedrick M N, Lonsdorf A S, Shirakawa A K, et al. CCR6 is required for IL-23-induced psoriasis-like inflammation in mice. J Clin Invest. 2009; 119:2317-2329. Kaser A, Ludwiczek O, Holzmann S, et al. Increased expression of CCL20 in human inflammatory bowel disease. J Clin Immunol. 2004; 24:74-85. Reboldi A, Coisne C, Baumjohann D, et al. C-C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE. Nat Immunol. 2009; 10:514-523. Varona R, Cadenas V, Flores J, Martinez A C, Marquez G. CCR6 has a non-redundant role in the development of inflammatory bowel disease. Eur J Immunol. 2003; 33:2937-2946. Matsui T, Akahoshi T, Namai R, et al. Selective recruitment of CCR6-expressing cells by increased production of MIP-3 alpha in rheumatoid arthritis. Clin Exp Immunol. 2001; 125:155-161.).
It is widely assumed that differentiation of T cells into specialized memory subsets also involves the acquisition and stable expression of homing- and chemokine receptor repertoires, allowing tissue- or inflammation-specific trafficking of these subsets (Butcher E C, Picker L J. Lymphocyte homing and homeostasis. Science. 1996; 272:60-66.). However, some studies also report a considerable plasticity in the expression of homing receptors. Whether this also applies to the expression of CCR6 is not known. CCR6 expression can be de novo induced on TCR-stimulated naive T cells by a cocktail of pro-inflammatory cytokines in combination with TGF-β (Acosta-Rodriguez E V, Rivino L, Geginat J, et al. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells. Nat Immunol. 2007; 8:639-646.).
Increasing evidence has been provided in recent years that differentiation of T cells into distinct lineages with stable phenotypes and functions involves epigenetic regulation of critical effector molecules (Ansel K M, Lee D U, Rao A. An epigenetic view of helper T cell differentiation. Nat Immunol. 2003; 4:616-623. Zhu J, Paul W E. CD4 T cells: fates, functions, and faults. Blood. 2008; 112:1557-1569. Wilson C B, Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation. Nat Rev Immunol. 2009; 9:91-105.) or lineage-specific transcription factors such as Foxp3 in Treg (Floess S, Freyer J, Siewert C, et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007; 5:e38. Huehn J, Polansky J K, Hamann A. Epigenetic control of FOXP3 expression: the key to a stable regulatory T-cell lineage? Nat Rev Immunol. 2009; 9:83-89. Baron U, Floess S, Wieczorek G, et al. DNA demethylation in the human FOXP3 locus discriminates regulatory T cells from activated FOXP3+ conventional T cells. Eur J. Immunol. 2007; 37:2378-2389.). Only few studies provided evidence that molecules involved in trafficking are subject to epigenetic regulation in T cells (Scotet E, Schroeder S, Lanzavecchia A. Molecular regulation of CC-chemokine receptor 3 expression in human T helper 2 cells. Blood. 2001; 98:2568-2570. Syrbe U, Jennrich S, Schottelius A, Richter A, Radbruch A, Hamann A. Differential regulation of Pselectin ligand expression in naïve versus memory T cells: evidence for epigenetic regulation of involved glycosyltransferase genes. Blood. 2004; 104:3243-3248.) or cancer cells (Sato N, Matsubayashi H, Fukushima N, Goggins M. The chemokine receptor CXCR4 is regulated by DNA methylation in pancreatic cancer. Cancer Biol Ther. 2005; 4:70-76. Mori T, Kim J, Yamano T, et al. Epigenetic up-regulation of C-C chemokine receptor 7 and C-X-C chemokine receptor 4 expression in melanoma cells. Cancer Res. 2005; 65:1800-1807).
Baba et al. (in Baba, M., Imai, T., Nishimura, M., Kakizaki, M., Takagi, S., Hieshima, K., Nomiyama, H. and Yoshie, O. Identification of CCR6, the specific receptor for a novel lymphocyte-directed CC chemokine LARC J. Biol. Chem. 272 (23), 14893-14898 (1997)) describe the identification of CCR6; Database entry NP_004358 discloses the amino acid sequence of human chemokine (C-C motif) receptor 6.
Rubie et al. (in Rubie, C., Oliveira, V., Kempf, K., Wagner, M., Tilton, B., Rau, B., Kruse, B., Konig, J. and Schilling, M. Involvement of chemokine receptor CCR6 in colorectal cancer metastasis Tumour Biol. 27 (3), 166-174 (2006)) propose an association between CCL20/CCR6 expression in human colorectal cancer and the promotion of colorectal liver metastasis. For this, 30 human cancer samples from colorectal tissue, 30 human samples from colorectal liver metastases and the adjacent nontumorous liver tissues were screened using quantitative real-time PCR, Western blot analysis, histochemistry, microdissection and the enzyme-linked immunosorbent assay (ELISA). While an overexpression of all the chemokine receptors was found in CRC, in colorectal liver metastases only the chemokine receptors CXCR4 and CCR6 were significantly upregulated.
WO 03/014153 describes CCR6 as similar to a cellular virus receptors and methods of use for said receptors.
A context between methylation of CCR6 and or BLR1 and certain types of immune cells, in particular T cells, has not been described.
Even though almost all cells in an individual contain the exact same complement of DNA code, higher organisms must impose and maintain different patterns of gene expression in the various tissue types. Most gene regulation is transitory, depending on the current state of the cell and changes in external stimuli. Persistent regulation, on the other hand, is a primary role of epigenetics—heritable regulatory patterns that do not alter the basic genetic coding of the DNA. DNA methylation is the archetypical form of epigenetic regulation; it serves as the stable memory for cells and performs a crucial role in maintaining the long-term identity of various cell types.
The primary target of methylation is the two-nucleotide sequence Cytosine-Guanine (a ‘CpG site’); within this context cytosine (C) can undergo a simple chemical modification to become 5-methyl-cytosine. In the human genome, the CG sequence is much rarer than expected except in certain relatively dense clusters called ‘CpG islands’. CpG islands are frequently associated with gene promoters, and it has been estimated that more than half of the human genes have CpG islands (Antequera and Bird, Proc Natl Acad Sci USA. 90:11995-9, 1993).
Aberrant methylation of DNA frequently accompanies the transformation from healthy to cancerous cells. Among the observed effects are genome-wide hypomethylation, increased methylation of tumour suppressor genes and hypomethylation of many oncogenes (reviewed by Jones and Laird, Nature Genetics 21:163-167, 1999; Esteller, Oncogene 21:5427-5440, 2002; Laird, Nature Reviews/Cancer 3:253-266, 2003). Methylation profiles have been recognised to be tumour specific (i.e., changes in the methylation pattern of particular genes or even individual CpGs are diagnostic of particular tumour types) and there is now an extensive collection of diagnostic markers for bladder, breast, colon, oesophagus, stomach, liver, lung, and prostate cancers (summarised by Laird, Nature Reviews/Cancer 3:253-266, 2003).
Epigenetic control by methylation is essential for early development including embryogenesis, X-chromosome inactivation and imprinting (monoallelic silencing) of either the paternal or maternal allele (Erlich, J Cellular Chem 88:899-910, 2003). There is also a class of genes that is active in the germ line, but is silenced by methylation in somatic cells (Bird, Genes and Dev 16:6-21, 2002; Li, Nature Reviews/Genetics 3:662-673, 2002).
Tissue-specific methylation also serves in regulating adult cell types/stages, and in some cases a causal relationship between methylation and gene expression has been established. The following is a partial list of genes, for which methylation changes are strongly implicated in controlling gene expression in tissue-specific manner: Lactate dehydrogenase C (testes); Oxytocin receptor (blood & liver); Tyrosine aminotransferase (liver); GFAP (astrocytes); and Leukosialin (leukocytes). In other cases, methylation may be a by-product of some other primary regulation, or it is required to lock the gene in the “off” state (Erlich, J Cellular Chem 88:899-910, 2003). For the present applications (immune cell identification(s)), a causal (biological) relationship is not required, but merely a strong correlation between methylation patterns and cell types.
A previously published example for such a cell type and cell status specific modification of certain gene regions is found during the lineage commitment of T cells to helper T cells (Th1 or Th2). Naïve (unstimulated) CD4+ T cells become activated upon encountering an antigen and can be committed to alternative cell fates through further stimulation by interleukins. The two types of helper T cells show reciprocal patterns of gene expression; Th1 produces Interferon-gamma (IFN-γ) and silences IL-4, while Th2 produces IL-4 and silences IFN-γ (Ansel et al., Nature Immunol 4:616-623, 2003). For both alternative cell fates, the expression of these genes is inversely correlated with methylation of proximal CpG sites. In Th2 and naïve T cells the IFN-γ promoter is methylated, but not in Th1 cells where IFN-γ is expressed (Attwood et al., CMLS 59:241-257, 2002). Conversely, the entire transcribed region of IL-4 becomes demethylated under Th2-inducing conditions, which strongly correlates with efficient transcription of IL-4. In Th1 cells, this extensive demethylation does not occur, rather particular untranscribed regions gradually become heavily methylated and IL-4 is not expressed (Lee et al., Immunity 16:649-660, 2002). Furthermore, Bruniquel and Schwartz (Nat Immunol. 4:235-40, 2003) have demonstrated that in naive T cells, the IL-2 promoter is heavily methylated and inactive, but after activation of the naïve T cell, the IL-2 gene undergoes rapid and specific demethylation at six consecutive CpGs. This alteration in methylation patterns occurs concomitantly with cell differentiation and increased production of the IL-2 product.
It is an object of the present invention to provide an improved method of expression analysis and in particular expression analysis based on DNA methylation analysis of the genes of the proteins CCR6 and/or BLR1 as a superior tool that can supplement or replace conventional methodologies as an indicator of cell type and status in vertebrates, in order to reliably identify certain immune cells, preferably T cells and/or B cells, of a mammal and/or in a mammal, in particular for a detection and quality assurance and control thereof.